SNARE Complex Assembly in Retinal Bipolar Neuron Exocytosis

Abstract

Three kinetic components of exocytosis have been described in retinal bipolar neurons. They are thought to reflect the fusion of a docked pool of ribbon-tethered synaptic vesicles termed the rapid pool, the releasable pool of ribbon-tethered vesicles, and a cytoplasmic reserve pool. In neurons, assembly of SNARE proteins facilitates exocytosis. We asked whether these pools could be distinguished on the basis of SNARE complex formation. Syntaxin3B is a t-SNARE in ribbon synapses. We generated a fluorescent peptide based on the syntaxin3B SNARE binding motif from goldfish. The peptide was dialyzed into isolated synaptic terminals of goldfish retinal bipolar cells via a whole-terminal recording electrode. A scrambled peptide served as control. Exocytosis was monitored with membrane capacitance measurements. Beginning one minute after break-in, a 1s stimulation, sufficient to deplete the releasable pool, was given every 60 seconds. The first exocytotic response was not significantly altered by the syntaxin3B peptide. However, by the fourth pulse, the exocytotic response in terminals dialyzed with the syntaxin3B peptide was reduced by 89% relative to the first, whereas that with the control peptide was reduced by only 45% (p < 0.04). This effect was not due to a reduction in calcium influx. Next, we implemented a pulse train protocol that captures the three components of release. Control terminals showed both depletion and replenishment of the pools. Terminals dialyzed with the syntaxin3B peptide showed: 1) immediate loss of the exocytotic component attributed to the reserve pool 2) decreased refilling of the rapid and releasable pools. The results demonstrate that reserve pool vesicles are unlike those in the rapid and releasable pools. It is likely that pool refilling is inhibited by the syntaxin3B peptide because reserve vesicles establish new SNARE complexes when they join a fusion-competent vesicle pool.